NK‐active cytokines IL‐2, IL‐12, and IL‐15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)‐2, IL‐12, and IL‐15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus‐infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL‐2, IL‐12, and IL‐15 on PKCα and PKC?—a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCα and PKC? activities; 2) whereas PKC? activity is induced by cytokine stimulation, PKCα activity is inhibited; and 3) both the induction of PKC? and the inhibition of PKCα functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine‐induced NK cell stimulation. Anat Rec 266:87–92, 2002. © 2002 Wiley‐Liss, Inc.     

Activation of protein kinase Cα signaling prevents cytotoxicity and mutagenicity following lead acetate in CL3 human lung cancer cells

Protein kinase C (PKC) family of serine/threonine protein kinases is sensitive signaling transducers in response to lead acetate (Pb) that could transmit phosphorylation cascade for proliferation and de-differentiation of neural cells. However, little is known as to the impact of PKC on Pb genotoxicity. Here we investigate whether Pb activates the conventional/classical subfamily of PKC (cPKC) signaling to affect cytotoxicity and mutagenicity in CL3 human non-small-cell lung adenocarcinoma cells. Pb specifically promoted membrane localization of the α isoform of PKC in CL3 cells. Pb also elicited Raf-1 activation as measured by the induction of phospho-Raf-1^S338 and the dissociation from the Raf-1 kinase inhibitor protein. Inhibition of cPKC activity using Gö6976 or depletion of PKCα by introducing specific small interfering RNA blocked the induction of phospho-Raf-1^S338, phospho-MKK1/2 and phospho-ERK1/2 in cells exposed to Pb. Intriguingly, declining PKCα enhanced the Pb cytotoxicity and revealed the Pb mutagenicity at the hprt gene. The results suggest that PKCα is obligatory for activation of the Raf-1–MKK1/2–ERK1/2 signaling module and plays a defensive role against cytotoxicity and mutagenicity following Pb exposure. Results obtained in this study also support our previous report showing that ERK1/2 activity is involved in preventing Pb genotoxicity.     

小鼠心室肌细胞容积敏感性Cl-电流的特性及调节（英文）

目的 :研究小鼠心室肌细胞容积敏感性 Cl- 电流的电生理学特性以及蛋白激酶 C （PKC）调节机制。方法 :膜片钳全细胞记录法记录分离的 C5 7BL / 6小鼠心室肌细胞低渗诱导的容积敏感性 Cl- 电流的变化。结果 :小鼠心室肌细胞存在典型的容积敏感性 Cl- 电流 ,其呈现外向整流性、高电位刺激下的时间依赖性失活、I- >Br- >Cl- 的可通透性和 Cl- 通道阻断剂 （DIDS）敏感的电生理学特性。而且 ,PKC激动剂 （PMA ）明显有助于该电流的激活。结论 :小鼠心室肌细胞的容积敏感性和外向整流性的 Cl- 电流受到 PKC激动剂的正向调节。     

Expression of Ca-dependent (classical) PKC mRNA isoforms after transient cerebral ischemia in gerbil hippocampus

Cerebral ischemia is known to modify the expression of genetic information in the brain. To complement this knowledge, in the present study we have estimated the expression of calcium- and phospholipid-dependent (classical) protein kinase C (c PKC) isoform mRNAs (α, βI and γ) at different time following ischemia. Forebrain cerebral ischemia was performed on Mongolian gerbils by 5 minutes bilateral occlusion of common carotid arteries. At the pointed time the cytoplasmic RNA was extracted from hippocampus and the expression of PKC mRNA quantified by RT PCR technique using GAPDH expression as an internal standard. Results indicate that only one γ isoform of cPKC mRNA expression becomes significantly modified in postischemic hippocampus. A transient increase up to 145% of control within the first 3 h was followed by its decline to 60–65% at a longer recirculation period. This lowered levels returned back to control at 72 h postischemic recovery. This result indicates that γ PKC could be particularly sensitive to ischemic insult and would react in accordance with the other early signals determining ischemic outcome.     

PKC在Ca2+信号调节MPF活性中的作用

用Fura-2AM作为染料,用F-2000荧光分光光度计测细胞内Ca2+浓度的改变。用A23187-Ca2+载体提高细胞内Ca2+浓度,同时用PKC的特异性抑制剂calphostin C作用细胞来阻断PKC途径,观察MPF活性改变。结果表明:300nmol/L的Calphostin C可以最大限度地抑制PKC活性。Calphostin C不能影响A23187引起的细胞内Ca2+浓度的升高。A23187处理前加入300nmol/L光激活Calphostin C不能降低MPF的活性,而A23187单独却可以引起MPF的活性降低。与对照组相比,差异不显著,p<0.05。     

神经系统S_(100)蛋白研究进展

S_(100)蛋白是钙结合蛋白。神经系统中主要为星形胶质细胞和雪旺氏细胞合成的S_(100β)。近年的研究表明它对神经元发育、轴突生长及突触可塑性等均有重要的作用。国际上近年对其生物学作用及作用机制研究活跃,本文就S_(100)的生物学特性、分布和来源、生物学作用及其作用机制等方面作一简介。     

妊马雌酮对大鼠体外培养成骨细胞作用的研究

目的 探讨妊马雌酮对大鼠体外培养成骨细胞的作用效果及对成骨细胞增殖的作用机理。方法 采用新生大鼠颅骨进行分离成骨细胞，并在含10％的胎牛血清DMEM中进行体外培养，分别加入高、低剂量妊马雌酮并与阴性组进行对照，用MTT法进行成骨细胞增殖和活性检测，用Western免疫印迹检测蛋白激酶C、抗凋亡蛋白Bag-1表达。结果 在培养24h后，妊马雌酮对培养成骨细胞增殖有明显的促进作用，培养到48h促进作用更为明显，表现出一定的时间相关性。妊马雌酮能明显提高体外成骨细胞PKC的表达量和抗凋亡蛋白Bag-1的表达量。结论 妊马雌酮可促进成骨细胞增殖并明显提高PKC和抗凋亡蛋白Bag-1的表达量是其防治骨质疏松的机理之一。     

Hypoxia-induced vascular endothelial growth factor expression in normal rat astrocyte cultures

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen, which also enhances vascular permeability. Because this angiogenic factor has been suggested to play a role in brain tumor biology, we have begun to investigate the regulation of VEGF expression in cultures of rat type I astrocytes. In this report, we have focused on the influence of hypoxia on VEGF expression. Under standard in vitro conditions (21% O₂) VEGF expression in astrocytes is barely detectable by northern analysis. However, after exposure to 0.2% O₂ for as little as 3 h VEGF mRNA levels are markedly increased reaching a maximum by approximately 8 h of exposure. Treatment of astrocytes with CoCl₂ or desferrioxamine results in a similar induction of VEGF, suggesting that the oxygen sensor regulating VEGF expression in astrocytes is a heme-containing molecule. Although acute treatment with TPA (6 h) induces VEGF expression, chronic exposure to TPA (24 h) to deplete PKC activity does not reduce the hypoxia-induced VEGF expression. These data indicate that VEGF induction in astrocytes can proceed through PKC-dependent and -independent pathways. Furthermore, chronic exposure to TPA or treatment with herbimycin A results in the enhancement of the hypoxia-mediated increase in VEGF mRNA levels. These results suggest that PKC and herbimycin-sensitive tyrosine kinase may serve as negative regulators of the hypoxia-activated signal transduction pathway that leads to the induction of VEGF expression. However, treatment of astrocytes with the nonspecific kinase inhibitors H7 and H8 reduced the level of VEGF induction by hypoxia, indicating that some type of kinase activity is required in this signaling pathway. © 1995 Wiley-Liss, Inc.     

Co-activation of insulin-like growth factor-I receptors and protein kinase C results in parasympathetic neuronal survival.

We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin-like growth factor-I (IGF-I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival-promoting activity. In combination with PdBu, however, IGF-I or insulin, at concentrations sufficient to act through the IGF-I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)-induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF-I. When added alone, aFGF-induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF-I, or insulin with PdBu under serum-free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF-I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.